| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
-Fibrinogen Subunit Gene from Xenopus laevis
Department of Physiology University of Missouri School of Medicine Columbia, Missouri 65212
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-fibrinogen subunit
gene from the frog Xenopus laevis. Transfection of cloned
DNA fragments into primary Xenopus hepatocytes showed that
the DNA between 163 and 187 bp upstream of the transcription initiation
site is essential for hormonal activation. A single complex forms when
this small region of DNA is incubated in vitro with
Xenopus liver nuclear proteins. The protein recognition
site has been narrowed to AAGAGTTAA, a sequence not previously
described as a transcription factor-binding site. We have named the
protein(s) bound to this sequence Xenopus glucocorticoid
receptor accessory factor (XGRAF). In addition to the XGRAF-binding
site, glucocorticoid regulation of the -fibrinogen gene requires at
least three nearby GREs, each of which is a poor match to the consensus
GRE. The position of the binding site for XGRAF overlaps the putative
upstream half of the most important GRE. Models are presented to show
possible ways that the novel accessory factor and the glucocorticoid
receptor could act through closely juxtaposed sites on the DNA. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The DNA sequence bound by GR is called a glucocorticoid response element (GRE). The consensus sequence for the GRE, GGTACAnnnTGTTCT, was derived from functional binding elements in multiple genes (1). GR can bind in vitro as a monomer to the downstream half of the GRE (TGTTCT), but transcriptional activation requires that GR interact with the full GRE as a dimer (9).
Glucocorticoids regulate hepatic production of several proteins as part of the acute phase response, a reaction to tissue injury, infection, or inflammation (10, 11). One of these proteins is fibrinogen, the precursor to fibrin, which is the primary structural protein of a blood clot. We have used a liver cell culture system from Xenopus laevis to examine mechanisms of transcriptional regulation of the genes coding for fibrinogen. This model is used because the fibrinogen genes from Xenopus have a strong steroid hormone response (12, 13).
Fibrinogen is secreted from the liver as a hexameric molecule
comprising two each of three different subunits, A
, Bß, and .
In Xenopus primary hepatocytes, glucocorticoid treatment
coordinately stimulates synthesis of the subunit mRNAs (12) through an
increase in transcription of each of the three genes (13). For the
Bß-subunit gene, a single GRE is located upstream of the
transcription initiation site (14). Mutation of this GRE eliminates the
ability of the Bß-fibrinogen gene upstream regulatory region to
respond to glucocorticoids. In the present report we examine
glucocorticoid regulation of the gene coding for the -subunit of
X. laevis fibrinogen.2
Rather than a single GRE as in the Bß-subunit gene, glucocorticoid
regulation of the -fibrinogen subunit gene requires accessory factor
binding to a novel DNA element and GR binding at multiple GREs.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-Subunit of
Xenopus Fibrinogen and Determination of the DNA
Sequence-fibrinogen
subunit gene were isolated that together encompass more than 21 kb of
DNA. The genomic DNA sequence from base -1537 to base +52 is presented
in Fig. 1
. This DNA includes the transcription
initiation site, identified by primer extension (data not shown) as an
A (+1 in Fig. 1) after a C, conforming to the consensus sequence
derived for transcription initiation sites (15).
|
-fibrinogen subunit gene was
connected to the luciferase reporter gene in the plasmid pLuc-Link 2.0
(pLL). The plasmid constructs were transfected into Xenopus
primary hepatocytes, which allowed the evaluation of the hormone effect
in a more physiological system than provided by transformed cell lines.
Luciferase levels were measured after 44–52 h in culture either
without hormone treatment or with 10-7 M
dexamethasone. The plasmid containing 5000 bp of the -subunit gene
upstream sequence responded to glucocorticoid treatment with an
approximately 3.5-fold increase in transcription (Fig. 2). This response was less than the 5- to 15-fold
stimulation observed for the -fibrinogen gene in run-on assays (13).
The discrepancy between the two assays is probably due to differences
in cell physiology. The run-on assays were performed in cells
maintained in culture for several days before hormone treatment, during
which time the cells become more responsive to glucocorticoids (our
unpublished observations). The transfected hepatocytes were cultured
only 2 days because the electroporation must be performed on freshly
isolated cells in suspension. Nonetheless, the 3.5-fold hormonal
stimulation observed in the transfection experiment is a
physiologically significant induction and permits analysis of unique
mechanisms responsible for regulation of the Xenopus
-fibrinogen gene by glucocorticoids.
|
-construct showed
that the 3.5-fold induction was maintained with as few as 187 bp of
upstream sequence (Fig. 2
). With further deletion to position -163,
the glucocorticoid response was severely decreased to only 1.4-fold.
Thus, the 24 bases encompassed by nucleotides -187 to -164 were
essential for glucocorticoid induction. The basal level of
transcription in the absence of hormone treatment was approximately the
same in all the deletion constructs containing from 105-5000 bp of
upstream sequence (see legend to Fig. 2). In particular, basal activity
did not change significantly when the hormone-responsive region from
-187 to -164 was removed.
The specific bases important for hormone regulation of -fibrinogen
gene expression were defined more precisely using site-specific
mutations (Fig. 3A) made in the -232 deletion construct
(pLL-232). A transfection construct with mutations in bases -181 to
-178 had a glucocorticoid response similar to wild type (Fig. 3B, mut
A). Mutation D, with base changes from -175 to -173, significantly
decreased the hormone induction (1.7-fold; Fig. 3B, mut D). A more
extensive mutation, from -182 to -173, impaired glucocorticoid
induction to the same extent (Fig. 3B, mut B). Mutation of bases from
-168 to -166 decreased the hormone response to only 1.3-fold (Fig. 3B, mut I). All three of these mutations (D, B, and I) affect the
sequence of a potential GRE from -177 to -163, AAGAGTnnnTGTTCC,
designated GRE1 (see Fig. 7). The downstream half of GRE1 has a strong
match (five of six positions) to the GRE consensus sequence. Mutation
I, which changed bases in the downstream half of GRE1, probably caused
loss of function by disrupting the GRE. The role of GRE1 and other GREs
in glucocorticoid regulation of the -gene will be discussed below.
Mutations B and D changed nucleotides in the upstream half of GRE1, but
this region was already a poor match to the consensus GRE. Therefore it
is unlikely that the decreased glucocorticoid response from the
constructs with mutations B and D was due to disruption of the
GR-binding site. These observations raised the possibility that a
non-GR transcription factor binds to these bases and is necessary for
glucocorticoid regulation.
|
|
-gene in
vitro. We used a crude extract from nuclei of Xenopus
hepatocytes that is enriched for transcription factors but is not
expected to contain a significant amount of GR (our unpublished
observations). When this extract was incubated with a DNA fragment of
the -gene from -200 to -115, a single DNA-protein complex was
detected in the gel shift assay (Fig. 4, lane 2).
Addition of 100-fold molar excess of nonlabeled double-stranded
oligonucleotide (33 bp from -189 to -157) completely eliminated
binding (Fig. 4, lane 3). Thus, hepatocyte nuclear protein(s) bound to
a 33-bp DNA fragment that encompassed the glucocorticoid-responsive
region.
|
). An
oligonucleotide with changes in bases -181 through -178 (mutation A)
competed for binding (Fig. 4, lane 4) as effectively as wild type
sequence (Fig. 4, lane 3), showing that these four bases were not
required for binding. Mutation D (bases -175 to -173) destroyed the
ability of the oligonucleotide to compete for nuclear protein binding
(Fig. 4, lane 5), demonstrating the importance of the bases at these
positions for the DNA-protein interaction. Mutation I (bases -168 to
-166) did not inhibit the ability of the oligonucleotide to compete
(Fig. 4, lane 6), indicating that the bases required for binding of the
nuclear protein complex did not include -168 to -166. Together these
experiments show that the binding site for the nuclear factor could
extend from -177 to -169.
Since mutation I disrupted the potential GR-binding site in this region
(GRE1), but did not affect formation of the protein-DNA complex
detected in the gel shift assay, it is unlikely that the protein in the
nuclear extract was GR. We confirmed that this nuclear protein-DNA
binding did not have the same sequence specificity as a GR-GRE
interaction by competition experiments. Excess oligonucleotide
containing either the GRE from the Xenopus Bß-fibrinogen
subunit gene or the palindromic GRE (14) did not compete for nuclear
protein binding to the wild type DNA (Fig. 5A, lanes 4
and 5). Thus the protein bound to the wild type sequence with a much
higher affinity than to a strong GR-binding site, identifying it as a
protein distinct from GR.
|
-DNA from -189 to -157 for matches to
protein-binding elements by searching the TFD and TRANSFAC
databases. No striking similarities to known transcription
factor-binding sites were identified, despite allowing two mismatches
in the search. One possibly meaningful match was from -173 to -168
(GTTAAT), a perfect match to one half of the palindromic binding site
for the liver-specific transcription factor, HNF-1. Binding sites for
HNF-1 were used as competitors for nuclear protein binding in the gel
shift assay. Neither the HNF-1 site from the Xenopus
Bß-fibrinogen gene nor a rat Bß-fibrinogen HNF-1 binding site (14)
was able to compete, demonstrating that the complex was not due to a
protein bound specifically to the HNF-1 site (Fig. 5A, lanes 6 and
7).
A second potentially important sequence was from -176 to -170. This
sequence is a five of seven match to the binding site for the
ubiquitous transcription factor AP-1 (TGAGTCA) (16). Therefore, we
determined whether the protein-binding site was bound by AP-1 in the
Xenopus nuclear extract. When the 33-mer oligonucleotide
(bases -189 to -157) containing the nuclear protein-binding site was
used as a probe in the gel shift assay, a single band appeared (Fig. 5B, lane 2). This binding was competed by both 20- and 100-fold excess
of the same nonlabeled oligonucleotide (Fig. 5B, lanes 3 and 4). A
second competitor was used that contained the AP-1-binding site, which
was made in the -189 to -157 oligonucleotide by changing position
-176 from A to T and position -171 from T to C. The AP-1
oligonucleotide provided little competition at 20-fold, but some
competition at 100-fold (Fig. 5B, lanes 5 and 6), indicating that the
Xenopus nuclear protein bound with much lower affinity to an
AP-1 site than to the -fibrinogen gene glucocorticoid-regulatory
region. To establish further that the protein bound to the
-fibrinogen hormone-responsive region was not AP-1, the
oligonucleotide that contains the AP-1 site was labeled and used as a
probe in the gel shift assay. The AP-1-specific probe was bound by
several proteins in the nuclear extract, none of which migrated at the
same position as the protein that bound to the wild type -fibrinogen
DNA (Fig. 5B, compare lane 8 to lane 2). Proteins bound to the AP-1
site were competed by 20- and 100-fold self-competition (Fig. 5B, lanes
9 and 10), but were not competed by 20- or 100-fold excess of the wild
type -fibrinogen DNA (Fig. 5B, lanes 11 and 12). Thus, proteins in
Xenopus nuclear extract that did bind an AP-1 site did not
bind the -fibrinogen sequence from -189 to -157.
The functionally important element from -177 to -169 appears to be a
novel transcription factor-binding site required for glucocorticoid
regulation of the -fibrinogen gene. We have named the protein(s)
bound to this site Xenopus glucocorticoid receptor accessory
factor (XGRAF).
GR-Binding Sites in the -Subunit Gene Upstream Region
Previously described accessory factors usually regulate
transcription in conjunction with nearby GREs (17). The region around
the XGRAF-binding site and downstream of the site was analyzed for
possible GREs by identifying GR-binding sites with the methylation
protection assay. The DNA upstream of -187 was not analyzed since the
5' deletion constructs indicated that no elements beyond -187 were
required for hormone induction (Fig. 2). Since the nuclear extract was
not likely to contain a significant amount of active GR, the protein
used for these assays was bacterially expressed GR DNA-binding domain
(GR-DBD), which is composed of amino acids 440–525 of the rat GR (20).
Only three amino acids near the C terminus of this section differ from
the comparable portion of X. laevis GR (21). The GR-DBD
exists as a monomer (22) and can bind either as a monomer to half-site
GREs or cooperatively as a dimer to GREs that have a high match to the
full consensus sequence (22, 23).
When GR-DBD was bound to the -upstream DNA, six guanines were
protected from methylation: on the sense strand at positions -167,
-155, and -139 and on the antisense strand at -164, -163, and -136
(Fig. 6, A and B). This protection pattern identified
three GR-binding sites that may be part of several potential GREs (Fig. 6C and Fig. 7). The protected guanines at -167, -164,
and -163 are within a binding site that could be the downstream half
of a GRE from -177 to -163 (GRE1, Fig. 6C). The guanines at -139 and
-136 are in a second binding site that could be the downstream half of
a GRE that extends from -149 to -135 (GRE3, Fig. 6C). GR-DBD binding
to GRE1 and GRE3 was only to the downstream halves, which match the
consensus well. No binding to the poor upstream halves occurred. The
protected guanine at -155 identified a third binding site that could
be part of either GRE2a from -165 to -151, or GRE2b from -156 to
-142 (Fig. 6C). The guanine at -152 on the antisense strand is also
part of a consensus match for GRE2a and GRE2b, but was not protected
(Fig. 6B). Therefore, the GRE2 area may have lower affinity for GR than
does either GRE1 or GRE3.
|
-232 construct
(Fig. 8).
|
, mut I=1.3-fold). This is lower than the response of
the construct with mutation D (1.7-fold), a mutation that eliminated
XGRAF binding (Fig. 4). Mutation I affected only GRE1, with no
consequence either on the XGRAF-binding site or on the matches to the
consensus sequence of the other potential GREs. Thus the large decrease
in the hormone response with mutation I showed specifically that the
downstream half of GRE1 was required for the glucocorticoid
induction.
|
, mut J),
indicating that either GRE2a or GRE2b was required for the full hormone
response.
As mentioned above, GRE3 was mutated in its upstream half by
mutation J, but the changes increased its match to the consensus by one
base so that GRE3 became a nine of 12 match to the consensus sequence
with a perfect downstream half. Therefore the decreased hormone
response caused by mutation J (Fig. 9, mut J) cannot be explained by
changes in the match of GRE3 to the consensus. The importance of GRE3
was analyzed with mutation L, which converted the downstream half of
GRE3 from a perfect match to only a one of six match. Overall, the
mutated GRE3 had only a three of 12 match to the consensus sequence.
Since mutation of these bases resulted in a 2-fold glucocorticoid
response (Fig. 9, mut L), GRE3 was necessary for the -subunit gene
to respond completely to glucocorticoids.
Analysis of the Region Encompassing Both the XGRAF-Binding Site and
GRE1
The XGRAF-binding site extends from -177 to -169 (Fig. 4) and
GRE1 is from -177 to -163 (Fig. 8). If glucocorticoid induction
requires binding of GR as a dimer to GRE1, then one monomer of GR must
interact with some of the same DNA sequence as XGRAF. We resolved which
protein was important for function at this position by examining both
protein binding and the hormone response with mutated binding
sites.
Both mutation B and mutation D changed bases in this potential overlap
region and reduced hormone responsiveness (Fig. 3). For mutation B the
reduction occurred despite the fact that GRE1 was made into a better
match to the consensus GRE. We expanded on this result with a new
series of site mutations that incrementally increased the match of the
upstream half of GRE1 to the canonical GRE (Fig. 10A). Mutation E matched three of
six bases in the upstream half. Mutation F matched the upstream half at
four bases, mutation G matched at five bases, and mutation C had a
perfect match of its upstream half to the GRE consensus. Each of these
DNAs contained the near perfect downstream half of GRE1. In addition,
each mutation changed bases in the XGRAF-binding site.
|
-fibrinogen DNA
(Fig. 10B, lane 2) was competed by an oligonucleotide containing the
XGRAF-binding site (Fig. 10B, lane 3). The four mutated
oligonucleotides that each had bases changed in the XGRAF-binding site
(Fig. 10A) were not able to compete for binding of the accessory factor
(Fig. 10B, lanes 4–7). Therefore, none of the mutated sequences were
able to bind XGRAF.
Since these mutations increased the match of GRE1 to the consensus, we
examined the ability of GR-DBD to bind the mutated sequences. In gel
shift assays the wild type DNA was weakly bound by only a monomer of
GR-DBD (Fig. 10C, lane 2). Probes containing mutations E, F, and G were
also bound by monomer of the protein, despite increasing matches to the
GRE consensus sequence (Fig. 10C, lanes 4, 6, and 8). In each case the
monomer probably bound to the wild type downstream half of GRE1 that
had five of six bases matching the consensus GRE. The DNA probe that
contained mutation C, with 11 of 12 bases that match consensus, was
bound by dimer of GR-DBD (Fig. 10C, lane 10). Therefore, none of the
mutations changed the ability of the DNA to bind GR-DBD except mutation
C, which created a stronger binding site.
The results of the binding experiments were compared with the effects
of the mutations on hormone inducibility. The wild type -232 deletion
construct had a 3-fold response to glucocorticoid treatment (Fig. 11). The construct containing mutation C had a
10.5-fold response (Fig. 11). This large hormonal induction, as well as
the binding of GR-DBD as a dimer to the DNA with mutation C (Fig. 10C),
are both probably due to introduction of a strong GRE that overrides
the wild type mechanism. Constructs with mutations E, F, and G had
decreased hormone responses compared with the wild type (Fig. 11).
Since each of the E, F, and G mutations did not alter GR binding (Fig. 10C), but did disrupt XGRAF binding (Fig. 10B), the loss of function
correlated with loss of XGRAF binding.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-fibrinogen gene requires multiple GREs and an accessory
factor binding site, all within the first 187 bp of the promoter. No
additional glucocorticoid-regulatory regions were found up to 5000 bp
upstream (Fig. 2), and no sequences with a strong match to the
consensus GRE were found within 1210 bases downstream (Genbank U66896).
The accessory factor site is a novel transcription factor recognition
sequence and is bound by a Xenopus liver nuclear protein
called XGRAF. The position of the XGRAF-binding element suggests that
it may be involved in a unique glucocorticoid-regulatory mechanism
since it overlaps the upstream half of the most critical GRE.
Fibrinogen Gene Regulation by Glucocorticoids
Our analyses of the Xenopus Bß- and -fibrinogen
genes are the only descriptions of specific elements required for
glucocorticoid regulation of fibrinogen genes from any animal. Although
glucocorticoids play a role in regulation of fibrinogen gene expression
in other species (25, 26, 27), the molecular mechanisms underlying the
induction have not been identified. Each of the rat and human
fibrinogen subunits for which the glucocorticoid response has been
examined has a general region of 150 to 1400 bases that confers
glucocorticoid regulation on the gene (28, 29, 30). However, no significant
matches to the consensus GRE were described for any of the genes in
either species. Thus, these genes may have complex
glucocorticoid-regulatory systems similar to the Xenopus
-fibrinogen gene.
Arrangement and Quality of GREs
We have identified several GREs within the Xenopus
-fibrinogen gene that are bound by GR in vitro and are
required for the functional response to glucocorticoids in
vivo. When examined as full GREs, the match between the sites in
the -gene and the 12 base consensus sequence ranges from five to
eight bases. These GREs could also be viewed as three tandem half-sites
(downstream halves of GRE1, GRE2a, and GRE3) that are similar to an
arrangement of half-site direct repeats reported to have a
glucocorticoid-responsive function in the MMTV gene (31). This isolated
region from the MMTV gene is the first description of glucocorticoid
induction mediated through half-site GREs arranged as direct
repeats.
Most glucocorticoid-regulated genes have one or more sites with a high match to the canonical GRE. Limited precedent does, however, exist for genes with functional GREs that have few matches to the consensus sequence. In the human alcohol dehydrogenase gene, ADH2, three GREs were identified by DNase I footprinting within a region required for glucocorticoid responsiveness (32). The best of these GREs has seven bases that match the 12-base consensus sequence, with only one of the four guanine moieties critical for binding (33). For the rat atrial natriuretic peptide gene, two positions were identified as GR-binding sites in a glucocorticoid-responsive region (34). Of these, the one with the highest similarity to the consensus sequence is only a seven of 12 match, with two of the four critical guanines.
Accessory Factors for Glucocorticoid Regulation
Several genes have been shown to require additional
DNA-binding proteins for complete regulation by glucocorticoids. The
rat tryptophan oxygenase gene is regulated through two GREs, but in
addition requires a CACCC box-binding element (35, 36). Regulation of
the rat phosphoenolpyruvate carboxykinase gene requires two DNA sites
that are bound by accessory factors in addition to two GREs. Multiple
proteins, including COUP-TF, HNF-4, and HNF-3, bind to these sites and
are required for the glucocorticoid response (3, 7). The rat TAT gene
has two glucocorticoid response units that involve HNF-3 and an Ets
family protein as accessory factors (4, 8, 37). The mouse proliferin
gene contains a composite GRE that includes an AP-1 site. Jun:Jun
homodimers are required for glucocorticoid induction, while Fos:Jun
heterodimers inhibit the hormone response (6). Glucocorticoid
stimulation of the stably transfected MMTV promoter requires a binding
site for the NF-1 accessory factor (5). All of these previously
reported accessory factor-binding sites are distinct from the
XGRAF-binding site upstream of the X. laevis -fibrinogen
subunit gene.
Models for XGRAF Interaction with the GR
An interesting aspect of the mechanism of glucocorticoid
regulation of the -fibrinogen gene is that the XGRAF-binding site
overlaps the putative upstream half of GRE1. We propose several
possibilities as to how these two factors can both be required at the
same binding position (Fig. 12). The first model
suggests that the two factors can bind to the DNA simultaneously. This
arrangement might be possible if the two proteins bind opposite sides
of the DNA helix. Since the DNA-binding domain of GR interacts with DNA
in the major groove (20), the minor groove could be accessible for
XGRAF binding.
|
The third model is that XGRAF binding displaces one part of the GR homodimer. In support of the feasibility of this model, the crystal structure of the GR-DBD was initially determined with a GRE containing one perfect half and one suboptimal half (20). The GR bound to this site as a homodimer through specific binding to the perfect half of the GRE and nonspecific binding to the other half of the site. XGRAF could stabilize the binding of GR homodimer in the absence of a GRE that has a high match to the consensus.
The fourth model requires sequential binding of GR and XGRAF. Two examples of this mechanism have been described. For the TAT gene the binding site for the accessory factor HNF-3 is in the same position as one of the GREs, and HNF-3 binding to this site is glucocorticoid-dependent (4, 8). In MMTV, glucocorticoid activation of transcription requires the accessory factor NF-1, for which binding to DNA is dependent on GR disruption of chromatin structure (5, 39).
The potential overlap between the XGRAF site and a poor GRE in the
Xenopus -fibrinogen gene may be part of a novel mechanism
for glucocorticoid action. Experiments are in progress to characterize
further the XGRAF protein and distinguish between the proposed models
of its action with GR. XGRAF or similar proteins may be involved in
glucocorticoid regulation of other genes, not only in
Xenopus but also in other species.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
fibrinogen subunit gene was cloned from
a X. laevis HD1 genomic library in 
gem11 provided by Dr.
Donald Brown of the Carnegie Institute. The 5'-portion of the
-subunit cDNA clone Xl3 (40) was used to screen the library
essentially as described (14) except that the bacterial strain was
ER1647 (New England Biolabs, Beverly, MA). The genomic DNA was digested
with restriction enzymes and subcloned into pBluescript SK-
(Stratagene, La Jolla, CA). The transcription initiation site was
identified by primer extension (41). The DNA sequence from -1537 to +1210 of both strands was determined on an Applied Biosystems Inc. (ABI, Foster City, CA) 373A automated sequencer using dye-termination methods as recommended by ABI. Sequencing was done at the Molecular Biology Program DNA Core Facility at the University of Missouri. Sequence information was analyzed for homology to known transcription factor-binding sites with the latest available versions of three different databases. The SITES table of TRANSFAC 2.5 (December 1995) (42) and the TFD database (release 7.5 SITES, March 1996) (43) were examined with the Findpatterns program of the Wisconsin Package, Version 8.0 (Genetics Computer Group, Madison, WI). The MATRIX table of TRANSFAC 3.0 (August 1996) (42) was searched with the program MatInspector 2.0 (44).
Reporter Gene Construction
Deletion Mutagenesis
The -fibrinogen gene deletion constructs -5000, -2900, -1158,
-603, -369, and -232 were made by restriction enzyme digestion of
genomic DNA subclones followed by ligation into the luciferase reporter
vector, pLuc-Link 2.0 (45). Often ligation of a short adapter sequence
or preliminary subcloning into pBluescript (pBS) was required. The
-105 deletion was created using Exonuclease III digestion, and
deletion constructs -187, -163, and -129 were prepared by PCR.
Primer oligonucleotides for PCR were obtained either from the Molecular
Biology Program DNA Core Facility at the University of Missouri or from
Genemed Biotechnologies (San Francisco, CA). Standard conditions of 10
µM primers, 4 ng template, and 2 mM
MgSO4 in an (NH4)2SO4
buffer (46) were used for all PCR with Taq enzyme.
Site-Specific Mutagenesis
The site-specific mutation construct made in pLL-187 (mutation I)
was prepared by PCR using Pfu polymerase (Stratagene, La
Jolla, CA) with the protocol from the manufacturer. DNA constructs with
mutations B, J, and L in pLL-232 were prepared by the megaprimer PCR
method (47). DNA constructs with mutations B and J were made using
Taq polymerase whereas that with mutation L was made with
Pfu polymerase (each with the conditions specified
above).
Constructs with mutations A, C, D, E, F, and G in pLL-232 were
produced by two-step PCR using Pfu polymerase with two
flanking primers and a pair of primers complementary to each other. One
of the complementary primers was used to make the mutation and upstream
DNA, while in a separate reaction the other complementary primer was
used to make the mutation and downstream DNA. The two products were
used in the second PCR as overlapping templates that primed on each
other.
For every construct the 3'-end of the -sequence is at the +41
position. Constructs were sequenced to ensure that both the 3'- and
5'-junctions were correct and that PCR products contained only the
desired mutations. Standard transformation protocols were used to
amplify the constructs in DH5 Escherichia coli (Bethesda
Research Laboratories, Rockville, MD) (48). Plasmid DNA was isolated by
alkaline lysis (49) followed by purification over an anion exchange
resin column (Tip 2500, QIAGEN, Chatsworth, CA). In addition, all DNA
used for transfection into liver cells underwent a single cesium
chloride gradient purification (48) to achieve optimal results in the
transfection assays.
Liver Cell Culture and Transfection and Assays for Reporter Gene
Activity
Adult female X. laevis (90–170 g) were treated with
estradiol (Sigma Chemical Co., St. Louis, MO) (14) to obtain
proliferating parenchymal cells (50). For each cell preparation,
primary cells were purified from the livers of two or three frogs by an
in situ perfusion method (14). Experiments were conducted in
accordance with the NIH Guide for the Care and Use of Laboratory
Animals. Transfection of freshly isolated hepatocytes was performed as
previously described with the modification that 50 µg luciferase
reporter DNA and 25 µg control plasmid pCMVßgal (51) were mixed
with cells for electroporation (14). Cells were incubated as before
with or without our standard hormone treatment consisting of
10-7 M dexamethasone and 10-9
M T3 (14). T3 is necessary for
optimal fibrinogen gene induction by glucocorticoids, but has no effect
by itself (12). After 48 h, cell extract was prepared using
nonionic detergent lysis (14).
Luciferase activity in the cell extracts was assayed as described (14) except the assay buffer included Coenzyme A at a final concentration of 700 µM (52). For the assay, 150 µl of cell extract were added to 540 µl of assay buffer and mixed briefly. Then 100 µl of 1 mM luciferin were automatically injected by the luminometer (Analytical Luminescence Laboratory, Westlake Village, CA; model 2010) and light production measured for 20 sec.
The activity of the ß-galactosidase produced from the control plasmid
was measured with the MUG assay (14) for some of the data in Fig. 2.
This assay was replaced by the Galactolight method (Tropix, Bedford,
MA) for the remainder of the data. Before either assay the endogenous
hepatocyte galactosidase was inactivated by heating the extracts at 48
C for 1 h (Tropix and Ref.53). For the Galactolight assay, 6 µl
cell extract diluted to 50% strength with lysis buffer were incubated
with 67 µl diluted Galacton substrate for 1 h, 100 µl
accelerator were injected, and light output was measured for 5 sec
after a 3-sec delay.
Nuclear Extract Preparation
X. laevis primary hepatocytes were isolated as
described (14) and incubated in the presence of 10-7
M dexamethasone and 10-9 M
T3 (Sigma) for 4–12 days at 2.5–4 x 105
cells/cm2 on Primaria substrate (Falcon, Franklin Lakes,
NJ), with one half of the medium replaced with fresh medium every 2
days. Nuclear extract was prepared by modification (54, 55) of Dignam
et al. (56). The cells (2–3 x 107) from
each flask were scraped from the substrate and washed three times with
ice-cold Barth (88 mM NaCl, 1 mM
K2SO4, 10 mM HEPES, pH 7.4), with
pelleting each time in a microcentrifuge at 13,800 x
g. All subsequent procedures were performed on ice in a 4 C
room. The cells were resuspended in buffer A+ (10
mM HEPES-KOH, pH 7.9, 10 mM KCl, 1.5
mM MgCl2, 0.1 mM EDTA, 0.1
mM EGTA, 1 mM dithiothreitol (DTT), 1
mM phenylmethylsulfonylfluoride (PMSF), 1 µg/ml
pepstatin, 5 µg/ml leupeptin, 3 µg/ml aprotinin, 0.05
mM benzamidine), pelleted at 13,800 x g,
and resuspended in 100 µl buffer A+ per 2.5 x
106 cells. NP40 was added to 0.3% (vol/vol), and the cells
were incubated for 10 min and centrifuged at 735 x g
in a microcentrifuge for 10 min to pellet the nuclei. The nuclei were
resuspended in buffer A+, pelleted at 13,800 x
g as before, resuspended in buffer C+ (10
mM HEPES-KOH, pH 7.9, 420 mM NaCl, 1.5
mM MgCl2, 1 mM EDTA, 1
mM EGTA, 20% glycerol, 1 mM DTT, 1
mM PMSF plus protease inhibitors as in A+
above) at 15 µl per 5 x 106 cells, and incubated
for 20 min. After centrifugation at 13,800 x g for 10
min, the supernatant was collected and buffer D (20 mM
HEPES-KOH pH 7.9, 50 mM KCl, 0.2 mM EDTA, 0.2
mM EGTA, 20% glycerol, 1 mM DTT, 1
mM PMSF) was added (3.75 volumes buffer D per 1 volume
supernatant). The nuclear extract was stored over liquid nitrogen.
Gel Shift Assays
Gel shift assays with purified rat GR DNA-binding domain
(GR-DBD) (20) were performed at room temperature as previously
described (14). Gel shift assays with hepatocyte nuclear extract
followed a protocol (57) modified to match the buffer and salt
conditions of the nuclear extract described above. For Figs. 4 and 5
the reactions contained in 14 µl final volume: nuclear extract (3
µg total protein in 5 µl), 2 µg poly(dI.dC), 3 µl of 5x
binding buffer (100 mM HEPES-KOH, pH 7.9, 250
mM KCl, 5 mM EDTA, pH 8, 50% glycerol, 0.5%
NP40, 5 mM DTT), and either no specific competitor or 20-
or 100-fold molar excess of specific competitor DNA (adding 15
mM NaCl). The reaction conditions in Fig. 10B were the same
except with 2 µl 5x binding buffer in a final volume of 9 µl.
Reactions were incubated 15 min on ice. Radioactively labeled probe in
1 µl was added, and the incubation was continued for 30 min on ice.
One-tenth volume of loading buffer (0.2% bromophenol blue and xylene
cyanol in 40% glycerol, 250 mM Tris-Cl, pH 7.5) was added,
and the free DNA was separated from the protein-bound DNA on a 5%
polyacrylamide (75:1 acrylamide-bisacrylamide), 0.1% NP40 gel using
0.25x TBE running buffer (22.25 mM Tris, 22.25
mM borate, 0.625 mM EDTA). The gel was run for
1.5–2 h and dried at 80 C for 2 h, and the DNA was detected by
exposure to XAR film (Kodak) at -80 C with one Lightning Plus
intensifying screen (DuPont, Wilmington, DE).
The -200 to -115 probe used for nuclear extract gel shifts was
produced by PCR with Taq enzyme. One of the primers used in
the reaction was 5'-end-labeled (49). The radioactively labeled PCR
product was purified on a native 6% polyacrylamide gel (48).
Oligonucleotides used for double-stranded probes or competitors were
purchased from the same sources as described above for PCR primers,
purified through a denaturing 16% polyacrylamide gel (48), ethanol
precipitated, and annealed (14). Those used as probes in Figs. 5B and 10C were end-labeled as before (49).
Methylation Protection Footprinting
Methylation protection probes extending from -232 to -6 or
from -232 to -42 were produced by PCR with Taq enzyme. For
selectively labeling either the top or bottom strand, the sense or
antisense PCR primer was 5' end-labeled (49). The PCR product was
purified through a native 6% polyacrylamide gel as above. The
methylation protection assays were performed according to a modified
procedure (58, 59) by incubating the labeled probe with 250 or 500 ng
GR-DBD in a 20-µl volume for 20 min under the conditions described
above for gel shifts with GR-DBD. The reactions were treated with 1
µl 10% dimethyl sulfate for 3 or 4 min at 22 C. Reactions were
stopped by addition of 180 µl of 10 mM Tris-Cl, pH 8, 0.1
mM EDTA, 50 µl of 1.5 M sodium acetate, pH 7,
1 M ß-mercaptoethanol, and 3 µl of 200 µg/ml yeast
RNA. After ethanol precipitation, the DNA was dissolved in 100 µl of
1 M piperidine and incubated at 90 C for 30 min, followed
by lyophilization to remove the piperidine. The DNA was twice dissolved
in 100 µl water and lyophilized before the products were analyzed on
a denaturing 6% polyacrylamide gel (49). The G + A sequencing ladders
were prepared by chemical sequencing methods (60).
|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
This work was supported by the following grants from the National Heart Lung and Blood Institute: RO1-HL-39095 (to L.J.H.), postdoctoral and predoctoral training grant HL-07094 (to M.L. and R.N.W.), and Research Career Development Award HL-02934 (to L.J.H.).
1 Present address: Gladstone Institute of Cardiovascular Disease,
University of California, San Francisco, San Francisco, California
94141. 
2 The genomic DNA sequence of the -fibrinogen
subunit gene from Xenopus laevis has been entered in
Genbank, accession number U66896.
Received for publication December 11, 1996. Revision received February 11, 1997. Accepted for publication February 14, 1997.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
, ß, and chains of rat fibrinogen following
defibrination. J Biol Chem 257:7277–7279 chain of human
fibrinogen. J Biol Chem 270:28342–28349 subunit of
Xenopus fibrinogen, the product of a coordinately regulated
gene family. Mol Cell Endocrinol 72:213–220[CrossRef][Medline]
B enhancer element. J Biol Chem 266:12722–12733This article has been cited by other articles:
 |
|
 |
|
  B. Morin, G. R. Woodcock, L. A. Nichols, and L. J. Holland Heterodimerization between the Glucocorticoid Receptor and the Unrelated DNA-Binding Protein, Xenopus Glucocorticoid Receptor Accessory Factor Mol. Endocrinol., March 1, 2001; 15(3): 458 - 466. [Abstract] [Full Text]
|
|
|
|
 |
|
 M. Li, X. Ye, R. N. Woodward, C. Zhu, L. A. Nichols, and L. J. Holland Analysis of the DNA-binding Site for Xenopus Glucocorticoid Receptor Accessory Factor. CRITICAL NUCLEOTIDES FOR BINDING SPECIFICITY IN VITRO AND FOR AMPLIFICATION OF STEROID-INDUCED FIBRINOGEN GENE TRANSCRIPTION J. Biol. Chem., April 17, 1998; 273(16): 9790 - 9796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Stoltz, M.-H. Vachon, E. Trottier, S. Dubois, Y. Paquet, and A. Anderson The CYP2B2 Phenobarbital Response Unit Contains an Accessory Factor Element and a Putative Glucocorticoid Response Element Essential for Conferring Maximal Phenobarbital Responsiveness J. Biol. Chem., April 3, 1998; 273(14): 8528 - 8536. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
